巴尔通体效应蛋白BepC与宿主p53蛋白互作诱导细胞凋亡机制研究

Bartonella is the pathogen of human bacillary angiomatosis. Studies had confirmed that Bartonella can induce the significant apoptosis of macrophages in lymph nodes of infected patients, which was thought to be the prerequisite of persistent infection and bacteremia. We speculated Bartonella type 4 secretion system (T4SS) effectors were the potential candidates causing apoptosis. Therefore, we transfected all the effectors of Bartonella quintana and Bartonella henselae in 293T cell to explore the cytotoxic protein and the global landscape of protein-protein interactions in host cells. Results confirmed BepC caused significant apoptosis in 293T and HeLa cells. Pull-down and MS/MS assays identified that BepC-induced cytotoxic effect depends on the binding with p53 protein, because BepC transfection in Jukat and CaCo (p53 mutation cell line) was unable to produce cytotoxicity. In this study, we aim to further investigate the mechanism of BepC-induced apoptosis in association with p53. The localization of BepC and p53 in transfected cells will be analyzed. Effect of BepC on p53 stabilization will be studied by western blot, and the transcription of p21 and MDM2 directly regulated by p53 will also be investigated after BepC transfection. In order to confirm the binding domains on p53, TAD,OD,DBD domains of p53 fused with Flag tag will be co-transfected with BepC fused with Strep tag, and will be detected by western blot. Furthermore, Fic and BID domians of BepC will be transfected in host cell to determine the domain causing apoptosis. Finally, the potential posttranslational modification of BepC on p53 will be analyzed by tandem MS/MS using the purified p53 protein that modified by BepC in vitro.

已有研究证实巴尔通体能诱导淋巴结内的巨噬细胞凋亡，导致感染宿主长期带菌并出现持续性菌血症，但其机理尚未明确。我们在前期研究中证实巴尔通体BepC蛋白通过与细胞p53蛋白结合能够诱导宿主细胞凋亡。为进一步揭示巴尔通体致细胞毒性因子及其作用机制，本项目将通过免疫荧光及蛋白杂交等技术研究BepC蛋白对p53蛋白含量、功能及细胞定位的影响；通过共转染实验分析BepC在p53蛋白上的结合位点（主要功能区TAD，OD，DBD）；并将BepC所含Fic及BID结构域分别转染细胞，确定其致细胞凋亡功能区；最后通过质谱分析BepC蛋白对p53蛋白潜在的翻译后修饰作用及位点（Fic结构域催化的腺苷酸化，AMPylation）,阐明巴尔通体BepC蛋白与细胞p53蛋白互作关系及促细胞凋亡机制，为巴尔通体免疫逃避及持续性感染机理提供新的科学解释。

本项目主要研究巴尔通体效应蛋白bepC对宿主细胞产生毒性作用的机理。bepC表达对宿主细胞产生小细胞分裂形式的毒性作用，表型与凋亡相似。通过蛋白蛋白互作关系证实bepC与细胞p53、HDAC1、SetD2等宿主核定位蛋白结合。首先bepC与HDAC1/2结合，导致组蛋白H3K36乙酰化显著降低，细胞核出现严重不稳定性，细胞在S期即出现核分裂现象。在HDAC1敲除细胞系中，bepC导致的核不稳定性被抑制。此外bepC与细胞三甲基化修饰酶setD2互作，导致细胞骨架与BepC共定位区域产生三甲基化修饰，并致细胞骨架皱缩出现小细胞分裂现象，bepC亦无法在setD2敲除细胞株中诱导细胞分裂毒性。通过突变体刷选实验发现，bepC与HDAC及SetD2的互作与bepC所含FIC结构域有关，Fic结构域点突变及敲除的bepC蛋白无法与HDAC及Setd2结合，亦无法导致细胞产生分裂毒性现象。通过对bepC本身在宿主细胞中产生的翻译后修饰鉴定发现，bepC存在多位点丝氨酸磷酸化修饰，其磷酸化水平与其Fic结构域有关；但本研究尚未成功鉴定BepC 对HDAC及SetD2的翻译后修饰类型。本项目研究发现了一种全新的细菌效应蛋白致细胞分裂毒性的表型，亦鉴定了Fic结构蛋白新的结合底物及潜在的新的翻译后修饰类型。尽管在项目执行期内我们获得了大量的新表型及新机制，但由于bepC原核表达蛋白的低活性问题，我们仍缺乏BepC 结合修饰底物及修饰类型的直接证据。课题组拟构建cas9全基因组突变细胞库，通过bepC导致的细胞毒性作用为筛选压力，进一步筛选确定bepC结合底物的直接证据，以期彻底阐明这种新的效应蛋白致毒性表型及机制。